Limb Girdle Muscular Dystrophy Type 2B (LGMD2B) and Miyoshi Myopathy (MM) are caused by mutations in dysferlin, but the role of dysferlin in healthy muscle and the changes that occur when it is mutated are still poorly understood. Although earlier studies suggested that dysferlin acts at the sarcolemma, our recent studies have yielded unexpected results: we find that dysferlin is selectively enriched in the transverse tubules (t- tubules: TT) of skeletal muscle. Challenging dysferlin null muscle with lengthening contractions in vivo or osmotic shock in vitro disrupts TT and causes Ca2+ channels to open, leading to significant changes in Ca2+ handling. In a recent FRET study, we obtained results to suggest that dysferlin and the dihydropyridine receptor (DHPR), the L-type Ca2+ channel that concentrates in TT at triad junctions (TJ), are located within 10 nm of one another in situ. Based on these preliminary results, we hypothesize that dysferlin protects muscle from injury-induced disruption of TT and dysregulation of Ca2+ by associating with DHPR and ryanodine receptors (RyR1) at TJ. We will test this hypothesis in experiments in healthy and dysferlin null muscles by studying the organization of TT, regulation of Ca2+ transients and baseline Ca2+ levels, and mechanochemical coupling between DHPR and RyR1. We will injure dysferlin-null and control muscles in vitro and ex vivo and muscle fibers in vitro and then apply ultrastructural approaches and state-of-the-art fluorescence-based imaging methods, including FRAP and FRET, to study the structural and functional changes in TT and TJ. We will test drugs that block L-type Ca2+ channels, stretch-activated channels, and ryanodine receptors, for their ability to protect dysferlin-null muscle from injury in vitro and in vivo. We will also prepare dysferlin variants, missing one or more of its structural domains or with point mutations linked to LGMD2B/MM, to identify the regions of dysferlin required to restore the structure and function of TT and TJ of dysf -/- muscle and to protect them from damage following stress. We have 3 specific aims: (1) to determine the role of dysferlin in regulating the function of TT and TJ; (2) to determine the role of dysferlin in regulating the structure and stability of TT and TJ; and (3) to identify the domains of dysferlin needed for it to concentrate in TT, interact with DHPR and RyR1 at TJ, and protect fibers from damage under stress. Our experiments will critically test our hypothesis that dysferlin protects TT structurally and functionally from damage by interacting with the two key components of TJ, DHPR and RyR1. They will elucidate the roles of dysferlin in skeletal muscle and how its absence leads to dysferlinopathy, and may suggest new avenues of treatment for LGMD2B/MM.